Purification of Thyrocalcitonin

Method 1 (R3). The procedure originally described for isolation of parathyroid polypeptides, parathormone in particular, requires the inclusion of cysteine as a reducing agent at two steps in the extraction procedure, since parathormone is sensitive to oxidation. Although there is no evidence that the hypocalcemic activity of thyrocalcitonin is affected by mild oxidation, cysteine has been retained in the thyrocalcitonin extraction procedure also. 

defatted thyroid powder is extracted with 8 M urea-0.1 M cysteine in 0.2 N HCl in the cold. Glacial acetic acid and acetone are then added, and the precipitate is filtered off and discarded. Diethyl ether is added to the filtrate, and the resulting precipitate is allowed to settle. After washing, the precipitate is dissolved in 20% acetic acid containing 0.01 M cysteine, and solid sodium chloride is added to give a concentra- 

Method 2 (Bl). This procedure, like that of Rasmussen, utilizes defatted thyroid powder obtained by repeated acetone washing of fresh pig thyroid tissue. This powder is then extracted with 0.2 A HCl at 60°C for 5 minutes, followed by 1 hour at room temperature. After filtering through cheesecloth, the filtrate is dialyzed for 24 hours against acetate 

Further purification of the salt-precipitated extract is achieved by passage through Sephadex G-lOO in 0.1 M acetate buffer (pH 4.6) containing 0.2 M NaCl. The main hypocalcemic activity is associated with the descending limb of the last main protein peak. A later communication from the same laboratory (G4) describes the use of 0.1 M formic acid in place of the acetate buffer for the Sephadex G-lOO stage. A further gel filtration step on Sephadex G-lOO in 0.1 M formic acid—1M urea-0.2 M NaCl is then used, and salt and other contaminants are removed by two or more runs on polyacrylamide beads. The active material is finally eluted as a single symmetrical peak. 

Additional purification of this relatively crude extract can be achieved by gel filtration on Sephadex G-50 equilibrated with 0.05 Af sodium acetate buffer (pH 3.8). Elution with the same buffer produces a large inactive protein fraction, followed by a smaller protein peak, and finally a nucleotide fraction. The hypocalcemic activity is associated with the descending limb of the second protein peak and the ascending limb of the nucleotide peak. The most active fractions are further purified by gradient elution with sodium acetate buffer on a carboxy-methyl-Sephadex G-25 column. Removal of the protein from the column is effected by gradient elution with sodium chloride in sodium acetate buffer. The hypocalcemic activity is associated with the descending limb of the protein peak. The authors claim that a 500-fold purification of thyrocalcitonin from the initial acid extract can be achieved. 

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